کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9604291 | 43615 | 2005 | 14 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer. Data concerning whole cell fluorescence and light scattering was collected. The whole cell fluorescence is probing intracellular concentrations of soluble fusion proteins. Concurrently, forward scattered light gives data about inclusion body formation, valuable information in process optimisation. To evaluate the method, the cells were disrupted, separated into soluble and non-soluble fractions and analysed by gel electrophoresis. A clear correlation between fluorescence and soluble target protein was shown. Interestingly, the distribution of the cells regarding forward scatter (standard deviation) correlates with the amount of inclusion bodies formed. Finally, the newly developed method was used to evaluate two different purification tags, His6 and Zbasic, and their effect on the expression pattern.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 119, Issue 2, 23 September 2005, Pages 133-146
Journal: Journal of Biotechnology - Volume 119, Issue 2, 23 September 2005, Pages 133-146
نویسندگان
My Hedhammar, Maria Stenvall, Rosa Lönneborg, Olof Nord, Olle Sjölin, Hjalmar Brismar, Mathias Uhlén, Jenny Ottosson, Sophia Hober,